The Effect of Ethanol on Cholesterol Crystals During Tissue Preparation for Scanning Electron Microscopy

Abstract

Dr. Abela and colleagues correctly point out that the use of ethanol, a dehydrating agent, in the preparation of the coronary thrombi samples for scanning electron microscopy may have dissolved cholesterol crystals and led to an underestimation of the true content of cholesterol crystals in the coronary thrombi retrieved by thromboaspiration. The study by Abela et al. (1) demonstrated that the use of vacuum dehydration of tissues is likely the preferred technique to study the impact of cholesterol crystals on biological membranes in vitro. In our study (2), we focused on the 2 main pharmacological therapeutic targets in acute coronary syndromes that can be visualized by microscopy, platelets, and fibrin fibers. Therefore, we preferred to use the ethanol dehydration technique instead of vacuum dehydration, to avoid the shrinkage and distortion of fibrin fibers that can occur with the latter technique. It was important to understand how the coronary thrombi evolve in time to understand the usefulness of treatments, such as fast-acting P2Y12 inhibitors or glycoprotein IIb/IIIa inhibitors, and their optimal use in the time course of the event. However, the hypothesis of cholesterol crystals perforating the intima and being the underlying mechanism of plaque rupture is seductive and deserves more in vivo studies. Unfortunately, no currently available technology can detect cholesterols crystals in vivo in patients experiencing acute coronary syndromes, and even though angioscopy has been used to define thrombus colors, the lack of magnification and 3-dimensional analysis as provided by scanning electron microscopy does not allow the identification of such small structures (3). Finally, it must be considered that the thromboaspiration technique does not allow a transversal analysis from 1 side of the artery to the other, as done in histological analysis of postmortem coronary arteries, but is most likely to retrieve a piece of the thrombus in the inner lumen, distant from the site of plaque rupture and therefore with fewer cholesterol crystals. This difference, added to the possibility of cutting-induced translocation of cholesterol crystals from the plaque itself to the inner lumen during tissue preparation, could be an explanation for the discrepancies in the amount of cholesterol crystals found in our study.