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Aim of the study The estimation of the upper reference limit (URL) for autoantibodies against thyroid peroxidase (TPOAbs) is a controversial issue, because of an uncertainty associated with the criteria used to correctly define the reference population. In addition, the URL of TPOAbs is method-dependent and often arbitrarily established in current laboratory practice. The aim of this study was to determine the reference limits of TPOAbs in a male sample according to the National Academy of Clinical Biochemistry (NACB) guidelines, and to compare them with those obtained in a female group, for five third-generation commercial-automated immunoassay (IMA) platforms. Methods 120 healthy males and 120 healthy females with NACB-required characteristics (younger than 30 years, TSH between 0.5 and 2.0 mIU/L, normal thyroid ultrasound, absence of thyroid disease and absence of other autoimmune diseases) were studied. Sera were analyzed for TPOAbs concentration using five IMA methods applied in automated analyzers: Immulite 2000 XPi (IMM); Maglumi 2000 Plus (MAG); Kryptor Compact Plus (KRY); Phadia 250 (PHA) and Liaison XL (LIA). Results A statistically significant difference (p