Detecting microcalcifications in atherosclerotic plaques by a simple trichromic staining method for epoxy embedded carotid endarterectomies

Abstract

Atherosclerotic plaques have a high probability of undergoing rapid progression to stenosis, becoming responsible of acute coronary syndrome or stroke. Microcalcifications may act as enhancers of atherosclerotic plaque vulnerability. Considering that calcifications with a diameter smaller than 10 mm in paraffin embedded tissue are rather difficult to detect, our aim was to analyze microcalcifications on semithin sections from epoxy resin embedded samples of carotid endarterectomies using an original trichromic stain (methylene blue--azur B--basic fuchsine--alizarin red). We have compared samples stained either with our method, methylene blue-azur B alone or with Von Kossa staining, and methylene blue-azur B -basic fuchsine alone or with Von Kossa staining. Our method resulted to be simple and fast (ca. 2 min), it gives a sharp general contrast for all structures and allows to easy identify collagen and elastin. In addition, gray-green colour associated to intracellular lipid droplets evidences foam cells, which are particularly abundant in endarterectomies samples. Mast cells and their metachromatic granules are also well recognized. Calcifications over 0,5 mm are clearly recognizable. In conclusion, microcalcifications are clearly distinguished from the extracellular matrix in spite of their reduced dimensions. Methylene blue--azur B--basic fuchsine--alizarin red method is easy to use, reproducible, and is particularly suitable for the identification of microcalcifications in the morphological analysis of atherosclerotic plaques.

Figure 1

Carotid endarterectomy. Monochromic staining. Boundary between thickened intima and media. The extracellular matrix appears in various cyan nuances. Cells are recognizable by dark blue nucleus and light blue cytoplasm. Elastin is clearly distinguishable from collagen only when organized within the internal elastic lamina (arrow), 400×.

Figure 2

Carotid endarterectomy. Monochromic staining. Boundary between atherosclerotic plaque fibrous cap and necrotic core. Monochromic staining does not allow distinguishing lipid vacuoles in foam cells' cytoplasm, being confused within the general ground colour. Microcalcifications are not clearly recognized; a large calcification is shown on the right side of the micrograph. Cholesterol clefts near the large calcification are not so easy to identify (arrow), 400×.

Figure 3

Carotid endarterectomy. Von Kossa staining followed by monochromic staining. The large calcification appears dark grey-black. Small calcifications are dark grey but they are very difficult to locate and to certainly identify as microcalcifications and not precipitates, because of the blue monochromic ground colour, 400×.

Figure 4

Carotid endarterectomy. Bichromic staining. Boundary between thickened intima and media. The use of this procedure enables a clear distinction between collagen (pink) and elastin (red violet), 400×.

Figure 5

Carotid endarterectomy. Bichromic staining. Boundary between atherosclerotic plaque fibrous cap and necrotic core. Contrast among different types of cells and the extracellular matrix is better than in monochromic staining, 400×. Microcalcifications are not so easy to detect if very small (<5 µm), because they appear very similar to collagen. Foam cells are clearly defined, with evident gray-green lipid vacuoles. Cholesterol clefts present near the large calcification are recognized (arrows), 400×.

Figure 6

Carotid endarterectomy. Von Kossa staining followed by bichromic staining. Large and small calcifications appear in dark rose. It is very difficult to discriminate such calcifications because they mingle with collagen (pink), 400×.

Figure 7

Carotid endarterectomy. Trichromic staining procedure. Boundary between thickened intima and media. The technique depicts a very similar image compared with Figure 4. In fact, collagen is stained in pink and elastin in purple, 400×.

Figure 8

Carotid endarterectomy. Trichromic staining procedure. Boundary between atherosclerotic plaque fibrous cap and necrotic core. Nuclei and cytoplasm are clearly distinguished. Foam cells show sharp boundaries and defined nuclear chromatin. The addition of alizarin red does not change the staining of lipid droplets, gray-green as in the bichromic procedure. Microcalcifications are simple to detect in spite of their dimension (clearly visible and identifiable if <2 µm), and are much highlighted by means of alizarin red. Cholesterol clefts present near the large calcification are visible (arrow), 400×. The large calcification appears more brilliant compared with Figures 3 and 6. This technique allows achieving a better contrast between microcalcifications and ground colour compared with Figures 5 and 6.

Figure 9

Carotid endarterectomy. Trichromic staining procedure. The contrast among all the elements of this trichromic image is improved. Small to very small calcifications are shown (the long arrow corresponds to the 0,5 µm calcification; the short arrow corresponds to the 2 µm calcification). The arrangement of fibres of the extracellular matrix is evidenced. Foam cells are clearly identified. Note the absence of precipitates, 1000×.