Histological evaluation disqualifies IMT and calcification scores as surrogates for grading coronary and aortic atherosclerosis Armelle Meershoek, Rogier.

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Histological evaluation disqualifies IMT and calcification scores as surrogates for grading coronary and aortic atherosclerosis Armelle Meershoek, Rogier A. van Dijk, Sander Verhage, Jaap F. Hamming, Antoon J. van den Bogaerdt, Ad J.J.C. Bogers, Alexander F. Schaapherder, Jan H. Lindeman International Journal of Cardiology Volume 224, Pages 328-334 (December 2016) DOI: 10.1016/j.ijcard.2016.09.043 Copyright © 2016 The Author(s) Terms and Conditions

Fig. 1 Movat Pentachrome (top) and Hematoxylin Eosin (H&E) staining (bottom) of atherosclerotic lesions in different aortic lesions. Representative images illustrating aspects of the Virmani classification system for atherosclerosis. Pathological Intimal Thickening (PIT) is characterized by presence of a an acellular lipid pool in the outer intima. Late Fibroatheroma (LFA) presents with a large necrotic containing multiple cholesterol crystals and that is covered by a fibrous cap. A Fibrous Calcified Plaque (FCP) represent represents a fibrotic lesion with a single, condensed calcified area [16]. Decalcification does not interfere with CCS scoring as footprints of earlier calcium deposits can easily be recognized in H&E staining (dark purple deposits) or Movat staining (brown and dark purple deposits in the detail (20-fold, right)). International Journal of Cardiology 2016 224, 328-334DOI: (10.1016/j.ijcard.2016.09.043) Copyright © 2016 The Author(s) Terms and Conditions

Fig. 2 Histological classification of human aortic atherosclerotic lesion type, and the relationship to age and sex. Three hundred and four aortic patches were characterized for the type of atherosclerosis according to Virmani et al. [16] and classified on basis of the highest degree of atherosclerosis present. The graph shows the highest grade of aortic atherosclerosis in relation to the age of the donor. There is a significant correlation of lesion type with age (r=0.640, P=0.01). Abbreviations; AIT: Adaptive Intimal Thickening, IX: Intimal Xanthoma, PIT: Pathological Intimal Thickening, EFA: Early Fibroatheroma, LFA: Late Fibroatheroma, TCFA: Thin Cap Fibroatheroma, PR: Plaque Rupture, HR: Healing Rupture, FCP: Fibrotic Calcified Plaque. International Journal of Cardiology 2016 224, 328-334DOI: (10.1016/j.ijcard.2016.09.043) Copyright © 2016 The Author(s) Terms and Conditions

Fig. 3 Histological classification of human coronary artery atherosclerotic lesion type and the relationship to age and sex. One hundred eighty five coronary samples were characterized for the type of atherosclerosis according to Virmani et al. [16] and classified on basis of the highest degree of atherosclerosis present. The graph shows the highest grade of aortic atherosclerosis in relation to the age of the donor. There is a significant correlation of lesion type with age (r=0.30, P=0.00005). Abbreviations are defined in the legend of Fig. 1. International Journal of Cardiology 2016 224, 328-334DOI: (10.1016/j.ijcard.2016.09.043) Copyright © 2016 The Author(s) Terms and Conditions

Fig. 4 Aortic IMT and CS versus the grade of atherosclerosis. Relations between histological IMT (the thick line represents the median IMT, dispersion around the median is shown by the 5th, 25th, 75th and 95th percentile lines). Presence of detectable (defined by presence of at least one or more calcium aggregates >200μm) is indicated by the robust dashed line; and the dominant aortic atherosclerotic lesion type). Abbreviations are defined in the legend of Fig. 2. International Journal of Cardiology 2016 224, 328-334DOI: (10.1016/j.ijcard.2016.09.043) Copyright © 2016 The Author(s) Terms and Conditions

Fig. 5 Coronary IMT and CS versus the grade of atherosclerosis. Relations between histological IMT (the thick line represents the median IMT, dispersion around the median is shown by the 5th, 25th, 75th and 95th percentile lines). Presence of detectable (defined by presence of at least one or more calcium aggregates >200μm in the lesion; indicated by the robust dashed line), and the dominant atherosclerotic lesion type. Abbreviations are defined in the legend of Fig. 2. International Journal of Cardiology 2016 224, 328-334DOI: (10.1016/j.ijcard.2016.09.043) Copyright © 2016 The Author(s) Terms and Conditions

Fig. 6 Instable lesions in calcified coronaries. A: Movat pentachrome staining showing a ruptured fibrous cap overlying a small calcified nodule (circle). Note presence of multiple lesions in this single cross section and the formation of a new lesion (neo atherosclerosis, greenish proteoglycan/collagen rich area, arrow) (12.5 fold (overview), and 100 times magnification (detail). The defect in the upper right corner reflects a cutting artifact. B: Movat Pentachrome staining showing a penetrating calcium nodule. Note the complete loss of media in the underlying area and an extremely thin, a-cellular overlying cap (12.5 fold (overview), and 40-fold magnification (detail)). Note: color legend for the Movat staining: blue: proteoglycans, yellow: collagen, black: elastin, red: smooth muscle cells and fibrinogen, purple: nuclei. Green reflects co-localization of proteoglycans and collagen. International Journal of Cardiology 2016 224, 328-334DOI: (10.1016/j.ijcard.2016.09.043) Copyright © 2016 The Author(s) Terms and Conditions