*4.3. Pathological Analysis*

Histological examination was performed by an experienced pathologist (I.C.). All adrenal glands included in the study were embedded in paraffin, cut into 3-μm-thick slices, and stained with hematoxylin and eosin (H&E). After accurate macroscopical and microscopical analysis, the final diagnosis of APA was established when a single nodule was present, while the final diagnosis of unilateral adrenal hyperplasia (UAH) was established in the presence of several nodules of varying sizes (with or without a dominant one). In the case of UAH, the dominant nodule was used for gene-expression studies, provided that it was the one identified with *CYP11B2* expression upon immunohistochemistry analysis.

After examination for the known features of ZF (large, lipid-laden clear cells with round to oval vesicular nuclei), ZG (small, compact cells with a high nuclear/cytoplasmic ratio, and a moderate amount of lipids), and zona reticularis (lipid-sparse cytoplasm, and compact cells) cells [20,30], the tumors were categorized as ZF-like when the percentage of large vacuolated cells was greater than 50%, and ZG-type when the percentage of ZF-like cells was ≤50% and the ZG-like cells were the prevalent cell type. Of the analyzed samples, 36 were previously included in the study by Monticone et al. [31].

The final histopathological diagnosis was APA in 35 cases, and multinodular hyperplasia in 11 cases. Twenty-five samples (23 APA samples and two UAH samples) were composed mainly of ZG-like cells, and 21 samples (19 APA samples and 3 UAH samples) were composed mainly of ZF-like cells.
