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.