Intraobserver and interobserver variability and spatial differences in histologic examination of carotid endarterectomy specimens

In this schematic representation of plaque segmentation, each transverse segment measures approximately 0.5 cm. The 0 segment, the reference segment that is routinely used for assessment of plaque histology in the Athero-Express study, is defined as the segment with greatest plaque burden. The adjacent segments at both sides are called +1, +2 etc, at one side and −1, −2, etc, at the other side.

Representative carotid plaque histology. Images A-G were photographed at ×20 original magnification. A, Hematoxylin and eosin (H&E) staining shows a plaque with no lipid core. B, H&E staining, large lipid core (L, lumen; C, core). The inset shows cholesterol crystals, which were clearly visible at higher magnification. C, Immunohistochemistry analysis with CD68 shows heavy macrophage infiltration. D, Immunohistochemistry analysis with α-actin shows heavy smooth muscle cell infiltration. E, Picrosirius red shows heavy collagen staining. F, Hematoxylin stains heavy calcification (blue). G, H&E, thrombus (arrows). H, At higher magnification (H&E, ×200 original magnification), signs of organization of the thrombus can be appreciated: angiomatosis (arrows) and influx of inflammatory cells.

Semiquantitative vs computer-aided measurements. Comparison of semiquantitative assessments, expressed as “no,” “minor,” “moderate,” and “heavy” (x-axis) and the computer-aided measurements, expressed as the percentage of plaque area occupied by the specific immunohistochemical staining in three representative fields (y-axis). A, Macrophage infiltration (CD68+). B, Smooth muscle cell infiltration (α-actin+).

Intersegment variability of histologic examination of the plaque. The similarity between different segments expressed as percentage of the κ value of the intraobserver repeatability, which is the theoretic maximum when two adjacent segments are completely equal: 100% indicates perfect similarity, 0% indicates no similarity, and negative values (not observed) indicate inverse associations. The similarity is compared between adjacent segments (intersegment distance “1”) and between the reference segment and a nonadjacent segment (intersegment distance “2”). MO, Macrophage infiltration; SMC, smooth muscle cell infiltration; coll, collagen; calc, calcifications; thro, thrombus; overall, overall phenotype.