*2.4. Anatomopathological Exam*

All 89 available hearts between 2009 and 2015 from our tertiary referral hospital with a heart transplant program were evaluated. Moreover, a patient diagnosed of LVNC by CMR was also transplanted. APE found eight hearts that fulfilled APE criteria for LVNC: six patients with isolated LVNC, one with congenital heart disease associated and one with concomitant three vessels ischemic disease.

The examination was performed by an experienced pathologist expert in the field, based on the LVNC anatomopathological criteria from Burke et al. [4,44]. Firstly, a macroscopic examination was performed. All hearts were systematically inspected, measured, weighted, and coronary sections were performed. They were examined for pathological changes in the four chambers, septum, pericardium, endocardium, and coronary arteries. Multiple samples were obtained, fixed in formaldehyde, paraffin embedded and stained with haematoxylin/eosin. Macroscopic findings were confirmed in microscopic sections. A minimum of three thin sections from each ventricle and two additionally from

the septal area were obtained from paraffin blocks. The macroscopic thickness was measured on the coronary sections of explanted hearts. We selected for microscopy the same area where the macroscopic measurement was performed, and then confirmed the measurements. Compaction and non-compaction wall thickness was measured in coronal macroscopic cuts and ratios were calculated and confirmed in haematoxylin/eosin samples. Histopathological exam was performed, studying fibrosis, inflammation, and cardiomyocytes' hypertrophy. All sections were stained with Haematoxylin-eosin, Masson trichrome, and Periodic Acid-Schiff (PAS) reaction. Fibre diameter measurements were performed only where the section produced a longitudinal view of cardiomyocytes. The measurement of each diameter was made at the nucleus height, and on a minimum of 50 fibres randomly selected. The nuclear size was measured systematically on longitudinal thinnest axis of nuclei, and over a minimum of 50, randomly selected. We used a Nikon microscope (Tokyo, Japan) with digital camera DS-FI2, and software—Nikon NIS D Elements (Tokyo, Japan), where annotations and measurements were registered. We performed all measurements with the same Nikon planacromatic objective size, using a scale provided from the software programme for each size of lens. The quantification was repeated twice in different journeys and performed by a pathologist and a technician. All the observations and results were reviewed by two professional cardiologists and pathologists.
