2.4. Histological analysis
The carotid atherosclerotic plaque was excised during CEA using a standard surgical technique with minimal and atraumatic manipulation of the specimen previously described by Sef et al. [29]. The carotid plaque specimen was obtained immediately after the surgical excision and then stored in fresh 4% paraformaldehyde solution at 4°C. The distal end of the plaque specimen was marked and processed by a standard procedure to obtain pathohistological specimen that includes fixation with 10% buffered formalin. The specimen was embedded in paraffin blocks, 4-μm-thick cross sections were dewaxed and prepared for subsequent staining.
The sections were stained with standard haematoxylin-eosin (H&E) and with Masson method for detecting collagen tissue which will be semiquantitatively analysed according to the study by Verhoeven et al. [30]. Each specimen was cut into 5-mm-long sections from the central part of the plaque resulting in a total of 5 sections for histological analysis. Plaque specimens were analysed for lipid, fibrous tissue and calcium. Lipid core area was expressed as a percentage of the total plaque area (TPA) as described previously using the following semiquantitative scale: 0= <10% of TPA, 1= 10-40% of TPA and 2= >40% of TPA [31, 32]. Histological examinations were performed independently by two experienced investigators blinded to the clinical history and MRI findings. Any discrepancies were resolved by discussion between the two investigators.