Results

One hundred eighteen frozen specimens from 45 3-vessel CABG patients were examined by standard histopathology. Fig. 4 is an example of Type III lesion. The thickened intima consisted of layers of foam cells underneath the endothelium (Fig. 4A). Figure 4B is the corresponding area of 4A, viewed by polarizing light at 10oC. The intima contained large amounts of lipid birefringence. When the sample was heated to 60oC, most birefringence disappeared (Fig. 4C) indicating that this lesion is rich in CE. The remaining birefringence was Cx. Fig. 4D is a digitized image of 4B. The lesion area border is indicated by a polygonal line. The lipid birefringence within the lesion was quantitated by selecting a threshold value T = 140. The ratio of the number of white pixels (lipid pixels) to the number of total pixels is a relative measure of the lipid amount in the lesion area. Fig. 4E is a digitized image of 4C. The cholesterol monohydrate crystal and phospholipid (PL) were quantitated by the same method (Fig. 4D). The amount of CE is the difference between the total lesion lipid (Fig. 4D) and Cx (Fig. 4E). This specific lesion contained 9.3% of total lipid, 6.6% of CE and 2.7% of Cx. No PL was detected in this lesion. The maximum intimal thickness was 346 µm.

Figure 4A Figure 4B
Figure 4C Figure 4D

Fig. 5 is an example of Type IV lesion. The intima had a very foamy appearance (Fig. 5A). The unstained section 5B, corresponding to 5A, contained a large amount of lipid birefringence when viewed by polarizing light at 10oC. At 60 oC, most birefringence remained (Fig. 5C) indicating that this lesion is rich in Cx. No obvious PL was found. The lesion total lipid and Cx were quantitated by the same method as Fig. 4. There were 44.1% of lesion total lipid, 38.7% of Cx and 5.4% of CE (Fig. 5D and 5E).

Figure 5A Figure 5B
Figure 5C Figure 5D,E

In summary, Fig. 4 lesion contains a smaller amount (9.3%) of lesion total lipid and is rich in CE (6.6%), and Fig. 5 lesion contains a larger amount (44.1%) of lesion total lipid and is rich in Cx (38.7%). These two demonstrations show that HSPLM and DIA can easily identify and quantitate a small amount (~3%) of lipid in the lesion (Fig. 4E) or a subtle change (~5%) of lipid birefringence (Fig. 5D, E).

The total lesion lipid and lipid composition of 118 samples were analyzed and shown in Fig. 6. Types II, III and IV lesions were rich in CE, and Type IV and Va were rich in Cx. PL was less than 1% in all the lesion analyzed, therefore Cx and PL was expressed as one measurement.

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