Several studies were conducted on the anti-pituitary antibodies (APA). APAs have been recognized for several years as the only molecular biomarkers for hypophysitis and were investigated with different techniques, such as the complement consumption test, immunoblotting with homogenate of human autopsy pituitaries, radioligand binding assays, and immunofluorescence [
13,
14]. Over the years, several attempts were made to optimize the immunofluorescence method, specifically to identify the best substrate. Experiments were conducted with pituitary slides from several animals: rats, rabbits, mice, baboons, and, eventually, humans [
15]. The baboon pituitary was considered the best substrate for APA identification. The serum APAs bind to the corresponding antigens present on the pituitary sections. The antigen-antibody complexes are detected by means of a goat anti-human IgG conjugated with a fluorescein isothiocyanate (FITC) [
3]. IgG FITC was adsorbed with monkey serum to remove non-specific fluorescence [
3]. The sera of patients were considered positive for a APAs starting at the dilution rate of 1:8 [
3]. The samples were considered positive in cases with a diffuse immunofluorescence pattern and an intracytoplasmic staining in the majority of the fields. In each assay, a positive and negative control needs to be included [
3]. The clinical relevance of APAs has been keenly discussed in previous research and APAs were widely considered a pathogenic marker of hypophysitis rather than a diagnostic tool. In fact, APAs were reported in other autoimmune disorders of the pituitary gland or in autoimmune systemic diseases, such as Sheehan’s syndrome, idiopathic growth hormone (GH) deficiency, idiopathic hyperprolactinemia, idiopathic hypopituitarism, brain traumatic injury, autoimmune polyendocrine syndromes and empty sella syndrome, but also in patients with pituitary adenomas or in healthy individuals [
14,
16,
17]. The experimental hypophysitis of SJL/J models showed that APAs may be detected with a higher concentration in the initial days after mouse immunization and gradually reduce thereafter [
11]. For these reasons, the APAs were also considered clinically helpful for the diagnosis of acute hypophysitis in humans, but only if detected at a high concentration [
16]. Recently, we proved that APAs are more prevalent in patients affected by PAH (68.4%) than in patients affected by not-secreting pituitary adenomas (22%) and in health controls (14%) [
18]. In the same study, we found that positivity for anti-pituitary and anti-hypothalamus antibodies was simultaneously detected in 52.9% of patients affected by PAH and in no patients carrying a non-secreting pituitary adenoma. As a consequence, although the presence of APAs may not exclude a non-secreting pituitary adenoma, the simultaneous positivity for anti-pituitary and anti-hypothalamus antibodies makes a diagnosis of not-secreting pituitary adenomas unlikely, with an odds ratio of 0.27 (95%IC: 0.13–0.57) [
18]. In addition, the detection of APAs positively predicts the outcome of treatment with glucocorticoids in PAH [
5].