Morphometric analysis of calcification and fibrous layer thickness in carotid endarterectomy tissues

Advanced atherosclerotic lesions are commonly characterized by the presence of calcification. Several studies indicate that extensive calcification is associated with plaque stability, yet recent studies suggest that calcification morphology and location may adversely affect the mechanical stability of atherosclerotic plaques. The underlying cause of atherosclerotic calcification and the importance of intra-plaque calcium distribution remains poorly understood.

The goal of this study was the characterization of calcification morphology based on histological features in 20 human carotid endarterectomy (CEA) specimens. Representative frozen sections (10μm thick) were cut from the common, bulb, internal and external segments of CEA tissues and stained with von Kossa׳s reagent for calcium phosphate. The morphology of calcification (calcified patches) and fibrous layer thickness were quantified in 135 histological sections.

Intra-plaque calcification was distributed heterogeneously (calcification %-area: bulb segment: 14.2±2.1%; internal segment: 12.9±2.8%; common segment: 4.6±1.1%; p=0.001). Calcified patches were found in 20 CEAs (patch size: <0.1mm(2) to >1.0mm(2)). Calcified patches were most abundant in the bulb and least in the common segment (bulb n=7.30±1.08; internal n=4.81±1.17; common n=2.56±0.56; p=0.0007). Calcified patch circularity decreased with increasing size (<0.1mm(2): 0.77±0.01, 0.1-1mm(2): 0.62±0.01, >1.0mm(2): 0.51±0.02; p=0.0001). A reduced fibrous layer thickness was associated with increased calcium patch size (p<0.0001).

In advanced carotid atherosclerosis, calcification appears to be a heterogeneous and dynamic atherosclerotic plaque component, as indicated by the simultaneous presence of few large stabilizing calcified patches and numerous small calcific patches. Future studies are needed to elucidate the associations of intra-plaque calcification size and distribution with atherothrombotic events.